...investigating any biological process. High quality genome sequence A high-quality sequence assembly of the ... Developmental biology insights using lineage tracing in zebrafish To highlight some of the features that...Lineage tracing studies highlight how these advantages can translate into insights in developmental biology...as a useful vertebrate model based on their straightforward husbandry and ability to produce abundant, ... a few of the available genetic resources and highlight two recent breakthroughs in developmental biology...lineage tracing approaches that led to important insights in developmental biology. Microscopy-based lineage... This microscopy-based lineage tracing defined a high-resolution map of early embryogenesis in time and...

...500nm) Light 0.1 Crystal Violet Pre and Post Visible Light 100 Methylene Blue Post Visible Light 100 ... and Post Blue or UV Light 0.5 GelRed Pre and Post (Post recommended) UV Light 0.1 GelGreen Pre and...you to visualize your DNA using UV light – please note that UV light can damage DNA and that proper personal...are having trouble getting colonies, you might want to use higher competency cells. Additionally, if your...bacteria; add chemicals to precipitate out the high molecular weight genomic DNA; filter the remaining plasmid...100 Ethidium Bromide Pre and Post UV Light 0.5 For more information on these stains see the Bitesize...final product is going to be very large (>10kb) you might want to use electro-competent cells instead of the...

...editing rates as high as 83% with NHEJ and 40-50% with HDR. Coselection efficiency is so high that some applications...Every quarter we highlight a subset of the new plasmids in the repository through our hot plasmids articles...27798609 Listen to our mCyRFP1 Podcast Segment New bright monomeric red fluorescent protein - mScarlet! The... new RFP variants for increased fluorescence, brightness, and proper maturation. Three new RFPs were isolated...toolbox Increasingly popular Optogenetics tools use light to alter cell physiology and molecular processes...protein is irreversibly cleaved when exposed to light. Robert Campbell’s lab illustrates the utility of...this caging technique to beautifully demonstrate light-dependent Gal4 transcriptional activation and Cre...

...from an mRNA that forms a pseudoknot. At high copy numbers, high levels of the lncRNA bind to RepZ mRNA ... termination. These types of plasmids tend to be high copy number. Here, we will consider the replication... ori called DnaA boxes. These boxes come in both high- and low-affinity versions, but binding of the low-affinity...: it bends the DNA to redistribute the DnaA from high-affinity boxes to low-affinity boxes, allowing lower... redistributes DnaA (red) from binding primarily high-affinity DnaA boxes (dark red) to also binding low-affinity...DnaA negatively regulates its own expression, so high levels of DnaA act as a feedback mechanism to reduce...stringently controlled plasmids, replication is tightly coupled to the bacterial host's cell cycle, maintaining...

... more carefully controlled. Regulated promoters might act only in certain tissues or at certain times ...them on or off at will with a chemical, heat, or light. In the cell, promoters themselves are controlled...of other promoter elements, and tend to be more tightly regulated. Common promoters for eukaryotes and ...Repressed by ethanol Full length version is strong with high expression. Truncated promoters are constitutive...promoter from maize ubiquitin gene Constitutive  Gives high expression in plants. H1 small RNA expression ...polymerase III RNA promoter Constitutive  May have slightly lower expression than U6. May have better expression...depending on its orientation to your gene. T7lac High levels of gene expression Promoter from T7 bacteriophage...

...species like C. elegans and Drosophila. Light inducible promoters Light is another way to activate gene expression...biology use light to regulate transcription. Red flame plasmid pDawn contains the blue-light sensing protein...switched from an OFF to an ON state, and how you might use these in your research. When you're done with...inducible promoters Chemical agents, temperature, and light are all examples of factors that can lead to the...protein YFI. When light is absent, YFI phosphorylates FixJ, which binds to the FixK2 promoter to induce... preventing expression of a reporter gene. When light is present, YFI is inactive, preventing repressor...transcription to take place. Addgene also has yellow flame light-regulated two component systems designed by the ...

... inducing the brightest fluorescence (Filonov et al., 2014). Broccoli is twice as bright as Spinach2 when...Mango fluoresces nearly as brightly as Spinach, but binds with much higher affinity to its cognate small... their high binding affinity to TO1-biotin. Two of these variants exhibit increased brightness compared...V., et al. "RNA mango aptamer-fluorophore: a bright, high-affinity complex for RNA labeling and tracking...biologist’s kitchen lab - and scientists using aptamers might know this best, as most fluorescent aptamers are...are 10-100 base nucleic acid oligos that bind with high affinity to small molecules to induce their fluorescence... the first aptamer identified to bind DFHBI, is highly unstable in vivo due to poor folding and thermal...

... variant has slightly different activation/inactivation kinetics. For example, the light-gated cation ...exists in several states: closed/light receptive, open, closed/light insensitive. If you deliver a continuous...to “reset” and become receptive to light once again. Therefore, light must be delivered in pulses. An example...the optogenetics coin is materials science-based. Light is delivered to your opsin through a small piece... optic cable implanted into the animal’s skull (right). The fiber optic cable is threaded through—and ...then connected from laser to ferrule to deliver light pulses to the target brain region. Thankfully, due...stimulation (the z-axis bregma coordinate). Because the light that is emitted from the end of the fiber optic ...

...the blue light molecule much, much, much more light-sensitive. Then you can use dim blue light and red ...the discovery of a light-sensitive opsin from algae that is sensitive to red light instead of blue, which...to tinker with brain circuits using the power of light. In a recent article published in Nature Methods...electrical activity of neurons on or off using pulses of light. By activating a neuron or set of neurons, you can...that all of these molecules are sensitive to blue light. Many have tried to make green molecules, but then...red light together. Understanding the brain through optogenetics research Addgene: What are some of the ... deep part of the brain, making it sensitive to light. When they illuminated this region, the animals ...

... not only brilliantly bright, but it also withstands denaturing conditions, high temperatures, and other...microscopy as well as correlative light and electron microscopy and observed high fluorescence resilience to ...retains folding and fluorescence in high molarity GdnHCl, at high temperatures, and in the presence of...Every few months we highlight a subset of the new plasmids, antibodies, and viral preps in the repository...CRISPR-Csm doesn’t degrade proximal RNAs nor does it have high levels of off-target effects, both of happen with...of antibodies we produced at Addgene and, as you might suspect, it has been one of our most commonly requested...8R] as another option for targeting GFP. But why might you need a second GFP antibody?  Similarities:  ...

...Signal Strength Lower Higher Sensitivity Higher Lower Background Lower Higher Post-translational ...bioluminescent reporter. This beetle emits a yellow-green light with a peak emission at 560nm. Shortly after the... which ideally would have low background signal, high sensitivity and of course be quick, accurate and...nature of the chemical reaction compared to using a high-intensity laser to rapidly excite GFP. As a result..., chemiluminescent reporters are generally less bright than fluorescent proteins, but have the advantage...Fluorescence Source of Emitted Photons Chemical reaction High-energy photons Kinetics of Photon Generation Slower...Subcellular Imaging Improving Well-established High-throughput assays Improving Well-established ...

...References 1. Viswanathan, Sarada, et al. "High-performance probes for light and electron microscopy." Nature methods...neuronal circuits and dynamics. FPs are generally bright, stable and well tolerated by cells. They can be...intrinsically fluorescent and can be detected with highly epitope-specific, fluorescently-labeled antibodies... signals or using anti-FP antibodies. GFP emits light in only a single channel. In multi-color imaging...the CNS. GFP and other FPs have the tendency to brightly label somata but not neuronal processes. In contrast...contrast, smFPs can label fine neuronal structures with high fidelity and at lower concentrations than GFP. smFPs...of these fine structures through their multiple, high-affinity binding sites for primary antibodies which...

...release. How is this light-mediated? The researchers split the proteolytically active light chain of BoNT/B...and SspB) that undergo light-induced dimerization. Upon application of blue light, iLID undergoes a conformational...Every few months we highlight a subset of the new plasmids in the repository through our hot plasmids...Listen to this episode of the Hot Plasmids podcast Light-mediated block of synaptic transmission Article ...podcast segment An optogenetic tool that allows light-mediated silencing of synapses was developed by ...conformational change allowing high affinity binding to SspB, brings together the split botulinum neurotoxin...created. The depositors demonstrated the utility of light-mediated suppression of synaptic transmission in...

...response and brightness. They also do not spectrally separate from green sensors and blue-light-activated...Physiol 2018. PubMed PMID: 30819783. Light up CreLite: a red-light inducible optogenetic tool for precision...Every few months we highlight a subset of the new plasmids in the repository through our hot plasmids...fluorescent protein (mScarlet) with either a seven or eight amino acid linker between them. The Ace-mScarlet...potential, Ace absorbance increases and results in higher quenching of mScarlet emission. Figure 2... with existing green-fluorescent GEVIs and blue-light-sensitive channelrhodopsins. Find the Ace-mScarlet...optogenetics. Cre activity is controlled by red light by splitting Cre and fusing each half with PhyB ...

...of painful responses in the left and right paw due to blue light activation and CNO silencing. (D) Immunohistochemistry...been made possible due to the collaborative and highly interactive environment that Janelia offers,” says...imaged 14 days post-transfection, excited at 505 nm (right). Scalebar represents 100 µm. Image from Abhi Aggarwal...expressed in the human cell line, Hep3B. From left to right: Mitotracker Red, GFP for the biosensor, Hoechst...changes following opioid withdrawal, providing insight into new methods for treating patients in recovery...involved in mechanical allodynia, a condition where a light touch creates a painful sensation. The dorsal horn...role in perceiving touch inputs. In this system, light activates the neurons (via oChIEF) and a chemogenetic...

...them to interact by stimulation with blue light.  Similarly, light can also be used to control homo-oligomerization...seconds after a pulse of blue light, and is reversible upon termination of light stimulation. CRY2clust enables...faster assembly and disassembly dynamics, higher sensitivity to light, and better distribution of clusters ...Every few months we highlight a subset of the new plasmids in the repository through our hot plasmids...oligomerization of target proteins in response to blue light. This tool, CRY2clust, was created by adding a 9...from Arabidopsis thaliana. When exposed to blue light, CRY2 undergoes a conformational change that permits...drawback of this method is the stray excitation of neighboring neurons in opsin dense regions. To overcome this...

...of the sensor, dLight1.1 and dLight1.2, exhibit high (submicromolar) DA affinity and high-magnitude responses...(Figure 2). The dLight1 dopamine sensor In 2018, Lin Tian’s lab has developed dLight1, a molecular tool... of DA release.  Using dLight1 sensors in experiments The Tian lab used dLight1 both in brain slices and...Feelings of delight associated with the brain’s reward circuits are generally linked to dopamine (DA)...Similar to calcium and glutamate sensors, the new dLight1 sensor is a single fluorescent protein-based sensor...learning and reward consumption in live mice Find dLight sensor here! The GRABDA dopamine sensor Yulong ...binding (Sun et al., 2018). These sensors also exhibit high magnitudes of fluorescent responses. Studies on ...

...to increase plasmid yield. Keep in mind that you might need to use multiple techniques for a particularly...additional steps are needed to optimize yield. You might want to include an extra wash step or use a different...recommends DH5𝝰! This strain consistently gives good, high quality preps due to its endA1 recA1 relA1 genotype...repeats. We use NEB Stable to transform and prep high quality DNA that contains these elements. Be aware...wash step to remove endonucleases (see figure at right for an example). Look out for carbohydrates Certain...purification use the cell’s resources to produce high levels of protein, not plasmid DNA. If you need ... increasing the culture volume, be aware that a higher volume of lysate passed over the binding matrix...

...Every quarter we highlight a subset of the new plasmids in the repository through our hot plasmids articles...articles. These brief articles highlight the main features and applications of a partiular plasmid or ...parallel analysis that successfully identified new, highly specific agonists for orphan GPCRs demonstrating...are found in one third of all cancers, including a high percentage (up to 95%) of pancreatic cancers. Ras...an additional 6 gRNAs per gene generated using slightly relaxed gRNA design parameters. To demonstrate...method in bacteria that enables rapid, automated, and high-throughput genome editing is multiplex automated...otherwise unmodified bacterial strains (5). MutL is highly conserved in distant relatives of E. coli so it...

...sometimes these fluorescent proteins might not localize to the right places, which can provide misleading...Every few months we highlight a subset of the new plasmids and viral preps in the repository through ...efficiency of tobacco etch virus protease (TEV), a highly sequence-specific protease used to cleave affinity...work for evolution of other target proteins and might be useful for your studies.  Find the plasmids to...including fusions with Histone 2A or 2B can result in bright targeted expression while minimizing non-specific...microtubules of mammalian cells, the author found that too high expression of these markers can lead to non-specific...biotin and biotin-4-fluorescein to be dissolved at a higher concentration, and results in uniform NB biotinylation...